Isolation of highly purified yeast nuclei for nuclease mapping of chromatin structure.
Probing chromatin structure with nucleases is a well-established method for determining the accessibility of DNA to gene regulatory proteins and measuring competency for transcription. A hallmark of many silent genes is the presence of translationally positioned nucleosomes over their promoter regions, which can be inferred by the sensitivity of the underlying DNA to nucleases, particularly micrococcal nuclease. The quality of this data is highly dependent upon the nuclear preparation, especially if the digestion products are analyzed by high-resolution detection methods such as reiterative primer extension. Here we describe a method to isolate highly purified nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the positions of nucleosomes at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the commonly used chromatin mapping and detection procedures.
Reese
Joseph C
JC
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA.
Zhang
Hesheng
H
Zhang
Zhengjian
Z
D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000737:chemistry*; D004271:DNA, Fungal / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism*; D009172:Mycology / Q000379:methods*; D011401:Promoter Regions, Genetic; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D002843:Chromatin; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C517042:RNR3 protein, S cerevisiae; D012262:Ribonucleoside Diphosphate Reductase; D008836:Micrococcal Nuclease